The major goal of the proposed studies is to evaluate humoral and cellular immune responses of colorectal cancer (CRC) patients to vaccinations (in Project 3) with recombinant adenovirus expressing the CRC-associated antigen GA733 (AdGA733-2). Evaluation of vaccinated patients' immune responses to AdGA733-2 will give insight into the immunological mechanism(s) that may underlie possible clinical effects of the vaccine. Both Epitope analysis of patients' immune responses will be carried out to identify GA733 antigen (Ag) fragments that may prove superior vaccine compared to the full-length Ag in future studies. To evaluate humoral immune responses in Ad-GA733-2 immunized CRC patients, induced antibodies will be tested for binding to GA733 Ag, Ag- positive tumor cells, cytotoxic reactivities against Ag-positive tumor cells and epitope specificity (using fragments of the GA733 Ag derived from Project 1). To evaluate T helper cell induction in Ad GA733-2 immunized patients, proliferative lymphocyte responses to the GA733 Ag will be demonstrated, followed by functional and phenotypic analysis of the proliferating cells. To evaluate cytotoxic T lymphocyte (CTL) responses in the vaccinated patients, CTL directed to the GA733 Ag will be identified. As an important correlate to the studies in CRC patients, the immune responses to the GA733 Ag- binding antibodies in approximately 50% of healthy individuals. Knowledge of the Ag-specificity and etiology of these immune responses is of utmost importance for the understanding of the mechanism(s) of immune response induction in CRC patients vaccinated with AdGA733-2. The proposed studies will determine the immunogenicity and mechanism of action of one of the best-studied CRC-associated Ags in patients. The identification of immunogenic epitopes/fragments of the GA733 Ag will provide new, potentially improved immunotherapies for CRC.